The aim of this proposal is to elucidate the mechanism of how WNK4 regulates Maxi K channel function. WNK (with no lysine (K)) kinase is a subfamily of serine/threonine kinases (5). Mutations in WNK1 and WNK4 result in pseudohypoaldosteronism type II (PHA II). PHA II, also referred to as Gordon syndrome, is an autosomal dominant disorder, characterized by hypertension, hyperkalemia, and metabolic acidosis (6). Emerging evidence indicates that WNK kinase contributes to a novel signaling pathway involving the regulation of ion transporters and channels that control sodium and potassium homeostasis. WNK4 WT was shown to inhibit renal outer medullary potassium (ROMK) channel activity and surface expression, whereas WNK4 PHA II- causing mutant further enhances the inhibition of ROMK activity (7). WNK1 WT was also shown to inhibit ROMK activity, whereas a kidney specific form of WNK1 (KS-WNK1) reverses the WNK1-mediated inhibition of ROMK (8). Maxi K, also referred as to BK or Slo, is the other major potassium channel in the distal nephron (9). Our preliminary data show that WNK4 WT not only significantly inhibits Maxi K channel activity and cell surface expression, but also reduces the total protein expression of Maxi K in mammalian cells. We also showed that WNK4 dead-kinase mutant D321A loses its inhibitory effect on Maxi K channel activity, indicating that the inhitory effect of WNK4 on Maxi K is kinase-dependent. We also found that WNK4 enhanced ERK1/2 phosphorylation, whereas knock-down of WNK4 reduced ERK1/2 phosphorylation. U0126, a MAPK inhibitor, reversed WNK4-mediated inhibition of Maxi K protein expression. These data indicated that WNK4 affects Maxi K activity and protein expression through a MAPK signaling pathway. We also showed that WNK4 WT interacts with Maxi K, whereas WNK4 dead-kinase mutant, D321A, retains interact with Maxi K while losing its inhibitory effect on Maxi K activity. However, it remains to be elucidated whether or not it is necessary for WNK4 to bind Maxi K in order to modulate Maxi K channel activity. The central hypothesis of this application is that WNK4 kinase modulates Maxi K activity either through affecting Maxi K protein processing or through altering the phosphorylation of Maxi K. To test this hypothesis, the following specific aims will be proposed: Specific Aim 1: Identify which domains of WNK kinase and Maxi K channel are responsible for their interaction and determine where they are colocalized in renal tubule cells and native tissue. Specific Aim 2: Determine the mechanism of the inhibitory effect of WNK4 on Maxi K protein processing. Specific Aim 3: Determine how WNK4 affects Maxi K activity through a phosphorylation dependent mechanism. Specific Aim 4: Determine the effects of dietary potassium changes and over-expressed WNK4 on renal potassium excretions and Maxi K protein abundances in mice. We will apply the methodologies of patch-clamp technique, cell biology, molecular biology, protein biochemistry and in vivo animal study to perform the proposed experiments. Further exploration of how WNK affects Maxi K channel function as well as the elucidation of the underlying pathophysiologic mechanism of PHA II caused by mutations of WNK kinases will provide a novel view on the regulation of the potassium channel activity and K secretion involving WNK kinase signaling in this research field. PUBLIC HEALTH RELEVANCE: Kidney plays an important role in the regulation of sodium and potassium homeostasis. WNK kinase is a novel serine/threonine kinase that represents a novel signaling pathway in the regulation of blood pressure and electrolyte homeostasis. Hypertension is one of most common diseases in the world. Elderly population including Veteran in USA has very high prevalence in hypertension. The aim of the present project is to investigate the role of WNK4 on Maxi K activity and elucidate the mechanism of WNK4-mediated regulation of Maxi K function. Further elucidating the mechanism of WNK4-mediated regulation of Maxi K and its relevant signaling pathway would provide a novel view on WNK4-mediated regulation of Maxi K activity and potassium balance.